Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 μm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and β-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.